U.S. patent application number 14/910401 was filed with the patent office on 2017-08-03 for novel use of fibroblast growth factor 2.
The applicant listed for this patent is BEIJING INSTITUTE OF MICROBIOLOGY AND EPIDEMIOLOGY. Invention is credited to Yueqiang DUAN, Chengyu JIANG, Chengcai LAI, Xin LIU, Xiliang WANG, Li XING, Penghui YANG.
Application Number | 20170216400 14/910401 |
Document ID | / |
Family ID | 52460464 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170216400 |
Kind Code |
A1 |
WANG; Xiliang ; et
al. |
August 3, 2017 |
NOVEL USE OF FIBROBLAST GROWTH FACTOR 2
Abstract
The present invention provides a novel use of fibroblast growth
factor 2 (FGF-2), i.e., a use of FGF-2 in preparation of medicine.
The uses of the medicine are the following (a) and/or (b) and/or
(c): (a) the prevention and/or treatment of lung injury; (b) the
prevention and/or treatment of influenza; (c) the prevention and/or
treatment of diseases caused by influenza viruses.
Inventors: |
WANG; Xiliang; (Beijing,
CN) ; JIANG; Chengyu; (Beijing, CN) ; YANG;
Penghui; (Beijing, CN) ; LIU; Xin; (Beijing,
CN) ; DUAN; Yueqiang; (Beijing, CN) ; XING;
Li; (Beijing, CN) ; LAI; Chengcai; (Beijing,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BEIJING INSTITUTE OF MICROBIOLOGY AND EPIDEMIOLOGY |
Beijing |
|
CN |
|
|
Family ID: |
52460464 |
Appl. No.: |
14/910401 |
Filed: |
August 9, 2013 |
PCT Filed: |
August 9, 2013 |
PCT NO: |
PCT/CN2013/000944 |
371 Date: |
February 5, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/50 20130101;
G01N 2800/12 20130101; G01N 33/6893 20130101; A61P 11/00 20180101;
A61K 38/1825 20130101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; G01N 33/68 20060101 G01N033/68 |
Claims
1-20. (canceled)
21. A method of protecting a mammal from a lung injury, an
influenza or a disease induced by an influenza virus or treating a
lung injury, an influenza or a disease induced by an influenza
virus in a mammal, said mammal being in need of said protecting or
treating, said method comprising administering FGF-2 to said
mammal.
22. The method according to claim 21, wherein the FGF-2 is a
human-derived FGF-2.
23. The method according to claim 22, wherein the FGF-2 is: (A) a
protein as set forth by SEQ ID NO. 1 in the Sequence Listing; or
(B) a protein derived from (A) with a substitution and/or a
deletion and/or an addition of one or more amino acid residues and
having the same activity as (A).
24. The method according to claim 21, wherein the mammal is in need
of the protecting or treating for a lung injury.
25. The method according to claim 24, wherein the lung injury is a
lung injury induced by a virus, a bacterium or a fungus.
26. The method according to claim 24, wherein the lung injury is a
lung injury induced by septicemia.
27. The method according to claim 21, wherein the mammal is in need
of the treating for influenza, or the protecting or the treating
for a disease induced by an influenza virus.
28. The method according to claim 27, wherein the influenza virus
is influenza A virus H1N1.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel use of fibroblast
growth factor 2.
BACKGROUND ART
[0002] The family of fibroblast growth factors (FGFs) includes 23
polymorphic growth factors with associated structures. Fibroblast
growth factor-2 (FGF-2), which is one of the members of the FGF
family, was extracted from bovine pituitary by American scientist
Gospodsrowicz D in 1974, and is widely present in the cells derived
from mesoderm and neural ectoderm as well as from various tumor
cells. It activates a FGF receptor on the membrane of a target
cell, mainly in an autocrine and/or paracrine manner, to induce a
series of intracellular signaling, and involves in various
physiological and pathological processes such as embryonic
development, angiogenesis, nerve regeneration, tumor growth,
etc.
[0003] FGF-2 has an isoelectric point, PI>9. 0, and is also
referred to as a basic fibroblast growth factor (bFGF). The gene of
FGF-2 is located in a human chromosome at 4q26, with a full length
of 38 kb, comprising 3 exons and 2 introns. The mRNA of FGF-2 has
multiple translation initiation sites, which can produce FGF-2
subtypes with various molecular weights, including subtype with a
low molecular weight of 18 kd and subtypes with high molecular
weights of 22, 22.5, 24 and 34 kd, but the FGF-2 with a low
molecular weight of 18 kd which comprises 155 amino acid residues
dominates. Low molecular weight subtypes are expressed in cytoplasm
and membrane of a cell, while high molecular weight subtypes mainly
directly enter into nuclei to function.
[0004] Acute lung injury (ALI) is an injury of alveolar epithelial
cells and capillary endothelial cells due to a variety of direct
and indirect injury factors, which can cause diffuse interstitial
and alveolar edema in lung, resulting in acute hypoxic respiratory
dysfunction. ALI is pathophysiologically characterized by reduced
lung volume, decreased lung compliance, and imbalanced ratio of
ventilation/blood flow; clinically characterized by progressive
hypoxemia and respiratory distress; and characterized in lung
imaging by inhomogeneous exudative lesions, called acute
respiratory distress syndrome as it develops into a severe stage
(oxygenation index<200). Common ALI-inducing factors are divided
into direct and indirect lung injury factors, wherein direct lung
injury factors include, for example, severe lung infection induced
by viruses, bacteria and fungi, aspiration of gastric contents,
lung contusion, oxygen poisoning, etc.; indirect lung injury
factors include, for example, sepsis, shock, massive blood
transfusion, cardiopulmonary bypass (CPB), disseminated
intravascular coagulation (DIC), etc.
[0005] Lung injury is clinically characterized by: (1) acute onset,
occurring within 12-48 hours after a direct or indirect pulmonary
trauma; (2) difficulties to correct hypoxemia after conventional
oxygen inhalation; (3) non-specificity of lung signs, hearable
moistrales or decreased breath sounds in both lungs at acute phase;
(4) early lesions, mainly interstitial, with no significant change
in chest x-ray film (C-XF), but occurrence of lung consolidation
after progression of disease, characterized by generally increased
density, decreased transparency, increased and thickened lung
markings, and visiable, discrete, patch-like, density-increased
shadows in both lung fields; (5) shadows of diffuse pulmonary
infiltrates, with no evidence of cardiac dysfunction.
[0006] Lung injury is clinically diagnosed through standards of:
(1) acute onset; (2) an oxygenation index
(Pa0.sub.2/Fi0.sub.2).ltoreq.200 mm Hg ((1 mm Hg=0.133 kPa,
regardless of positive end expiratory pressure (PEEP) level; (3)
patch-like shadows in both lungs shown in anteroposterior C-XF; (4)
pulmonary artery wedge pressure (PAWP) 18 mm Hg, or no clinical
evidence of increased pressure in left atrium. For example, it may
be diagnosed as ALI when showing Pa0.sub.2/Fi0.sub.2 300 mm Hg and
meeting other standards described above.
[0007] Lung injuries induced by respiratory viruses, bacteria, or
fungi are the most common acute respiratory infections in clinic.
Among these, influenza is a common and frequent disease affecting
extremely widely on people, and there is now a grim situation of
cross-species infection by influenza virus. Infection with
influenza A virus H1N1 leads to clinical symptoms which are
relatively mild in most patients, characterized in typical
influenza-like symptoms, and can be recovered naturally. The most
common symptoms include cough, fever, sore throat, headache, and
other discomforts. Severe pneumonia patients have visible multiple
lesion infiltration in C-XF, which can rapidly develop into ARDS,
kidney or multi-organ failure. The incidence of influenza A
combined with ARDS may be 100 folds of normal influenza. Lung
damages are primarily derived from uncontrolled systemic immune
response, and like ARDS that is secondary to viral pneumonia,
include diffuse alveolar damage, bronchiolar and perivascular
lymphocytic infiltration, hyperplastic airway changes, and
bronchiolitis obliterans.
[0008] Both of clinical and pathological examinations indicate that
serious patients may have lesions mainly in the respiratory system.
It can be seen from a pathological examination that serious
patients may have consolidation in lung, often accompanied with
pathological changes such as bleeding, effusion, abscessus, etc.
Serous effusion or fibrinous effusion found in alveolar space,
accompanied with varied degrees of transparent film formation,
which is indicative of diffuse lung injuries. It is currently
considered that the basic lesions of pulmonary tissue injuries
induced by influenza A virus H1N1 is similar to those of lung in
serious cases resulted from other types of influenza, SARS, RSV,
adenoviruses, parainfluenza, recently emerged SARS-like viruses,
Human avian influenza H7N9, etc, i.e., varied degrees of diffuse
pulmonary tissue injuries.
[0009] Lipopolysaccharides (LPSs), which are a group of
water-soluble and glycosylated lipoplexes, are important
ingredients in outer membrane of a gram-negative bacterium, and
formed of three parts of lipid A, core polysaccharide and antigen
O. LPS have a molecular weight of more than 10000 Daltons, with a
complicated structure. Lipid A is a glycolipid contributive to
endotoxin activity, covalently linked to a heteropolysaccharide
chain. Human is extremely susceptible to bacterial endotoxin, and
even a very small amount (1-5 ng/1000 g body weight) of endotoxin
can induce an increased body temperature, a fever reaction which
often lasts for about 4 hours and then gradually subsides. In the
case of a natural infection, because of continuous growth and
proliferation of gram-negative bacteria, accompanied with one after
another death and release of endotoxin, the fever reaction will
last until pathogens are completely eliminated in the body.
[0010] Fever reaction is induced by endotoxin because the endotoxin
acts on macrophages and the like in bodies to produce cytokines
such as interleukin-1, interleukin-6, and tumor necrosis
factor-.alpha., etc., which in turns act on the thermotaxic center
of hypothalamus in the host, resulting in increased body
temperature and fever. Endotoxemia has clinical symptoms mainly
depending on host's resistance to endotoxin, and the symptoms and
signs thereof can include: fever, a changed number of leukocytes, a
bleeding trend, heart failure, renal function insufficiency, liver
damage, neurological syndromes, and shock, etc. Endotoxin can cause
release of histamine, serotonin, prostaglandin, kinin, and the
like, leading to expansion of microcirculation, reduced volume of
venous return blood, decreased blood pressure, inadequate tissue
perfusion, hypoxia, acidosis, etc.
[0011] Fungi can also affect lung tissues and result in lung
injuries which may be mainly characterized by fungal inflammation
or related diseases in lung and bronchi, and possibly those in
pleura or even mediastinum. Pathogenic fungi belong to primary
pathogens, which often induce a primary exogenous infection in an
individual with normal immune function. Conditioned pathogenic
fungi, alternatively called opportunistic fungi, have low
pathogenicity, mostly inducing a deep fungal infection in a
susceptible host.
[0012] Zymosans are macromolecular polysaccharide complexes
extracted from yeast cell walls, formed of proteins and
carbohydrates. Zymosans can be used to induce inflammations in lab,
and the induced reactions thereby mainly include expression of
inflammatory cytokines, upregulation of arachidonic acid,
phosphorylation of partial proteins and formation of lipositol.
Moreover, zymosans are capable of upregulating the expression of
cyclin D2, which indicates that zymosans also play a role in the
process of activation and proliferation of macrophages.
[0013] The infection with LPS in combination with zymosan may be
used to simulate ALI induced by septicemia in vivo. Septicemia
refers to an acute systemic infection induced by a pathogen or a
conditioned pathogen which invades into blood circulation, and then
grows and proliferates in blood, thereby producing toxins.
Septicemia is one of risk factors of ALI, and one of the
characteristics of septicemia-induced lung injury (SLI) is
aggregation and activation of polymorphonuclear neutrophils (PMN)
in pulmonary microvassels, giving rise to a series of inflammatory
reactions and vascular injuries. In this process, bacterial
infection, particularly gram-negative bacterial infection may be a
key factor for initial inflammatory reaction. Gram-negative
bacteria and LPS, after entering into a circulation, produce a
LPS-binding protein (LBP), which will bind to a part of
phospholipid A of LPS. The LPS-LBP complex bind to CD14 receptors
on mononuclear cells, macrophages, and main neutrophils in plasma,
to facilitate the translation of coding genes of specific
inflammatory factors (such as TNF-a, IL-1, IL-6). The cytokines are
secreted into circulation, which is an important biochemical
characteristic in a series of inflammatory reactions responsible
for septicemia and lung injuries. These cytokines such as IL-1,
IL-6, IL-8, IL-10, IL-12, etc. will induce a series of cascade
reactions, and participate the process of lung injury. Therefore,
use of the infection with LPS in combination with zymosan may allow
for a simulation of septicemia-induced lung injury.
[0014] To this end, there is already a great and urgent need for
developing a new drug for the treatment and/or prevention of lung
injuries in the life sciences.
DISCLOSURE OF INVENTION
[0015] The present invention provides a new use of FGF-2.
[0016] The present invention provides a new use of FGF-2, i.e. a
use of FGF-2 in the manufacture of a drug for the use of (a) and/or
(b) and/or (c) as follows: (a) preventing and/or treating a lung
injury; (b) preventing and/or treating influenza;
[0017] (c) preventing and/or treating a disease induced by an
influenza virus.
[0018] The FGF-2 may be a human-derived FGF-2.
[0019] The FGF-2 may be the following (A) or (B): a protein as set
forth by SEQ ID NO. 1 in the Sequence Listing; (B) a protein
derived from (A) with a substitution and/or a deletion and/or an
addition of one or more amino acid residues and having the same
activity as (A).
[0020] The lung injury may be a lung injury induced by a virus
and/or a bacterium and/or a fungus. The virus may be an influenza
virus, particularly influenza A virus H1 N1, more particularly
influenza A virus H1 N1 BJ501 strain or influenza A virus H1N1 PR8
strain. The bacterium may be a gram-negative bacterium,
particularly Escherichia coli, more particularly E. coli 0111: B4.
The fungus may be a yeast, more particularly Saccharomyces
cerevisiae.
[0021] The lung injury may be a lung injury induced by
septicemia.
[0022] The lung injury may be a lung injury induced by LPS and
zymosan A.
[0023] The influenza may be influenza A, particularly an influenza
induced by influenza A virus H1N1, which may be particularly
influenza A virus H1 N1 BJ501 strain or influenza A virus H1 N1 PR8
strain.
[0024] The influenza virus may be influenza A virus H1 N1, more
particularly influenza A virus H1 N1 BJ501 strain or influenza A
virus H1 N1 PR8 strain.
[0025] The present invention also seeks to protect a drug having an
active ingredient of FGF-2; for the use of following (a) and/or (b)
and/or (c): (a) preventing and/or treating a lung injury; (b)
preventing and/or treating influenza; (c) preventing and/or
treating a disease induced by an influenza virus.
[0026] The FGF-2 may be a human-derived FGF-2.
[0027] The FGF-2 may be the following (A) or (B): a protein as set
forth by SEQ ID NO. 1 in the Sequence Listing; (B) a protein
derived from (A) with a substitution and/or a deletion and/or an
addition of one or more amino acid residues and having the same
activity as (A).
[0028] The lung injury may be a lung injury induced by a virus
and/or a bacterium and/or a fungus. The virus may be an influenza
virus, particularly influenza A virus H1 N1, more particularly
influenza A virus H1N1 BJ501 strain or influenza A virus H1N1 PR8
strain. The bacterium may be a gram-negative bacterium,
particularly E. coli, more particularly E. coli 0111: B4. The
fungus may be a yeast, more particularly S. cerevisiae.
[0029] The lung injury may be a lung injury induced by
septicemia.
[0030] The lung injury may be a lung injury induced by LPS and
zymosan A.
[0031] The drug may also comprise an additional active ingredient
which may be synergistic with the FGF-2. The drug may also comprise
a pharmaceutical substance such as a preservative, a stabilizer, a
buffer, and the like. The drug may be in a dosage form of an
injection, a spray, a nasal drop, an inhalant, or an oral
agent.
[0032] The influenza may be influenza A, particularly an influenza
induced by influenza A virus H1N1, which may be particularly
influenza A virus H1N1 BJ501 strain or influenza A virus H1N1 PR8
strain.
[0033] The influenza virus may be influenza A virus H1N1, more
particularly influenza A virus H1N1 BJ501 strain or influenza A
virus H1N1 PR8 strain.
[0034] The present invention also seeks to protect a use of FGF-2
in prevention and/or treatment of a lung injury. The FGF-2 may be a
human-derived FGF-2.
[0035] The FGF-2 may be the following (A) or (B): a protein as set
forth by SEQ ID NO. 1 in the Sequence Listing; (B) a protein
derived from (A) with a substitution and/or a deletion and/or an
addition of one or more amino acid residues and having the same
activity as (A).
[0036] The lung injury may be a lung injury induced by a virus
and/or a bacterium and/or a fungus. The virus may be an influenza
virus, particularly influenza A virus H1N1, more particularly
influenza A virus H1N1 BJ501 strain or influenza A virus H1N1 PR8
strain. The bacterium may be a gram-negative bacterium,
particularly E. coli, more particularly E. coli 0111: B4. The
fungus may be a yeast, more particularly S. cerevisiae.
[0037] The lung injury may be a lung injury induced by
septicemia.
[0038] The lung injury may be a lung injury induced by LPS and
zymosan A.
[0039] The present invention also seeks to protect a use of FGF-2
for preventing and/or treating influenza, or for preventing and/or
treating a disease induced by an influenza virus.
[0040] The FGF-2 may be a human-derived FGF-2.
[0041] The FGF-2 may be one of following (A) or (B): a protein as
set forth by SEQ ID NO. 1 in the Sequence Listing; (B) a protein
derived from (A) with a substitution and/or a deletion and/or an
addition of one or more amino acid residues and having the same
activity as (A).
[0042] The influenza may be influenza A, particularly an influenza
induced by influenza A virus H1N1, which may be particularly
influenza A virus H1N1 BJ501 strain or influenza A virus H1N1 PR8
strain.
[0043] The influenza virus may be influenza A virus H1N1, more
particularly influenza A virus H1N1 BJ501 strain or influenza A
virus H1N1 PR8 strain.
[0044] The present invention also seeks to protect a use of FGF-2
as a marker of a lung injury, or use of a substance for detecting
FGF-2 in aiding diagnosis of a lung injury, or use of a substance
for detecting FGF-2 in the manufacture of a product for aiding
diagnosis of a lung injury.
[0045] The FGF-2 may be a human-derived FGF-2.
[0046] The FGF-2 may be the following (A) or (B): a protein as set
forth by SEQ ID NO. 1 in the Sequence Listing; (B) a protein
derived from (A) with a substitution and/or a deletion and/or an
addition of one or more amino acid residues and having the same
activity as (A).
[0047] The FGF-2 is particularly the FGF-2 in serum, plasma, or a
lung lavage fluid.
[0048] The lung injury may be a lung injury induced by a virus
and/or a bacterium and/or a fungus. The virus may be an influenza
virus, particularly influenza A virus H1N1, more particularly
influenza A virus H1N1 BJ501 strain or influenza A virus H1N1 PR8
strain. The bacterium may be a gram-negative bacterium,
particularly E. coli, more particularly E. coli 0111: B4. The
fungus may be a yeast, more particularly S. cerevisiae.
[0049] The lung injury may be a lung injury induced by
septicemia.
[0050] The lung injury may be a lung injury induced by LPS and
zymosan A.
[0051] In the present invention, a FGF-2 gene knocked-out mouse
model and an influenza A virus H1N1 infected mouse model are used
to demonstrate the important role of FGF-2 in an acute pathological
injury of pulmonary tissues and death of a mouse induced by
infection with influenza A virus H1N1, and the important role of an
intervention targeting FGF-2 molecules in the treatment of a lung
injury, particularly of an injury induced by the infection with
influenza A virus H1N1. In the present invention, FGF-2 is used for
treating a mouse model infected with influenza A virus H1N1. The
results indicate that FGF-2 plays an important role in the
protection of a mouse from the acute pathological injury of
pulmonary tissues induced by the infection with influenza A virus
H1N1. Therefore, it is first demonstrated by the present invention
that FGF-2 plays an important role in the pathological process of
influenza A, and FGF-2 is capable of preventing or delaying the
serious consequences from infection with influenza A virus.
[0052] The present invention also uses LPS from a gram-negative
bacterium and zymosan A from a yeast to co-infect a mouse, and it
is found that an intervention targeting FGF-2 possibly plays an
important role in the treatment of the lung injuries induced by
infection of LPS from a gram-negative bacterium in combination with
zymosan A from S. cerevisiae. The present invention uses FGF-2 to
treat a mouse model co-infected with LPS and zymosan A, and as a
result, it is shown that FGF-2 can exert a significant protection
for mice from acute pathological injuries of pulmonary tissues
induced by infection with a composition of LPS and zymosan.
Therefore, it is for the first time demonstrated by the present
invention that a FGF-2 recombinant protein can prevent or delay the
serious consequences from co-infection with LPS and zymosan A.
DESCRIPTION OF DRAWINGS
[0053] FIG. 1 shows the results in Example 1.
[0054] FIG. 2 shows the slice staining results in Example 2.
[0055] FIG. 3 shows the wet/dry ratio results in Example 2.
[0056] FIG. 4 shows the statistical results of survival rate in
Example 3.
[0057] FIG. 5 shows the statistical results of body weight changes
in Example 3.
[0058] FIG. 6 shows the slice staining results in Example 3.
[0059] FIG. 7 shows the wet/dry ratio results in Example 3.
[0060] FIG. 8 shows the statistical results of survival rate in
Example 4.
[0061] FIG. 9 shows the slice staining results in Example 4.
[0062] FIG. 10 shows the wet/dry ratio results in Example 4.
[0063] FIG. 11 shows the slice staining results in Example 5.
BEST MODES TO CARRY OUT THE INVENTION
[0064] Following Examples are intended to facilitate better
understanding of the present invention, but not for limiting the
present invention. Those experimental methods used in the following
Examples are conventional methods, unless otherwise specified.
Those experimental materials used in the following Examples are
commercially available from a conventional biochemical reagent
supplier, unless otherwise specified. Those quantitative tests in
the following Examples each were conducted triplicate, with the
results averaged. The data is analyzed and processed with software
GraphPad Prism 5. In the statistics of survival rate, after being
infected with viruses, those mice which died in 24 h are considered
as non-specific death, and are excluded from the statistics of
survival rate.
[0065] C57 BL/6 mice (4-week old): Laboratory Animal Center of The
Academy of Military Medical Science. FGF-2 gene knocked-out mice
(with a background of SPF grade C57 BL/6 mice): American Jackson
Laboratory, Catalog No. 003256. LPS (LPS, from LPS of E. coli 0111:
B4): Sigma, L2630. zymosan A (Zymosan A, from S. cerevisiae):
Sigma, Z4250. FGF- 2 recombinant protein (Human recombinant
FGF-2/basic FGF protein): protein sequence is shown as SEQ NO: 1 in
the Sequence Listing, encoding gene thereof is shown as SEQ NO: 2
in the Sequence Listing; Millipore, Catalog No. 01-106, when used,
it is diluted into desired concentration with PBS buffer.
[0066] Influenza A virus H1N1 A strain /Beijing/501/2009 (H1N1)
(abbreviated as BJ501 strain):
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=info&id=6
488568&lvl=3&keep=1&srchmode=1&unlock&lin=s;
Yang P, Deng J, Li C, Zhang P, Xing L, Li Z, Wang W, Zhao Y, Yan Y,
Gu H, Liu X, Zhao Z, Zhang S, Wang X, Jiang C. Characterization of
the 2009 pandemic A/Beijing/501/2009 H1N1 influenza strain in human
airway epithelial cells and ferrets. PLoS One. 2012;7(9):e46184.
doi: 10.1371/journal.pone.0046184. Epub 2012 September 26.
[0067] Influenza A virus H1N1 influenza strain in human airwa
epithelial cells and ferrets. PLoS One.
[0068] Influenza A virus H1N1 A strain/Puerto Rico/8/1934 (H1N1)
(abbreviated as PR8 strain):
[0069]
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode32
Info&id=21
1044&lvl=3&lin=f&keep=1&srchmode=1&unlock; Li
C, Yang P, Sun Y, Li T, Wang C, Wang Z, Zou Z, Yan Y, Wang W, Wang
C, Chen Z, Xing L, Tang C, Ju X, Guo F, Deng J, Zhao Y, Yang P,
Tang J, Wang H, Zhao Z, Yin Z, Cao B, Wang X, Jiang C. IL-17
response mediates acute lung injury induced by the 2009 pandemic
influenza A (H1N1) virus Cell Res. 2012 Mar; 22(3):528-38. doi:
10.1038/cr.2011.165. Epub 2011 October 25.
[0070] The PBS buffer used in the Examples is 0.01 mol/L PBS
buffer, pH7.2, unless otherwise specified.
Example 1. Increased FGF-2 Level in Mouse Lung Lavage Fluid Induced
by Influenza A virus H1N1
[0071] For the experimental group (5 of 4-6 weeks old C57 BL/6
mice): each of the mice was securely fixed, and intraperitoneally
injected with 50-60 .mu.L of 1 g/100 mL solution of pentobarbital
sodium for anesthesia; the anesthetized mice were kept with their
head backward and upward leaned to bring their nasal cavities into
an upward position, and 10 .mu.L virus solution of BJ501 strain
(10.sup.5.5 TCID.sub.50/mouse) was added dropwise to their nasal
cavity at each side through a pipette; the mice were kept in this
posture for 15 seconds, to allow the virus to enter into their
respiratory tracts; 24 hours after the infection with BJ501 strain,
the mice were killed by means of intraperitoneal injection of
excessive anesthetic; the killed mouse was fixed on a small animal
operating table, and the skin and bone of its chest were removed, a
small opening was cut on its trachea, and 800 .mu.L PBS buffer was
injected into the mouse through the opening; finally, lung lavage
fluid (LLF) was aspirated by three repeated imbibitions, and
detected for the concentration of FGF-2 therein by a Bio-Plex Mouse
Cytokine 23-flex kit.
[0072] For the control group (5 of 4-6 weeks old C57 BL/6 mice):
the mice were treated in the same way as for the experimental
group, except that an equal volume of allantoic fluid of chick
embryo was used instead of the virus solution of BJ501 strain.
[0073] The results are shown in FIG. 1. The mice infected with
influenza A virus H1N1 have a FGF-2 concentration in LLF higher
than that of the control group (*P<0.05), that is, the
expression level of FGF-2 in LLF is significantly higher in the
mice infected with influenza A H1N1 than that in the control group.
The results indicate that FGF-2 plays an important role in
influenza A H1N1 induced lung injury, and an intervention targeting
FGF-2 can hold an important position in the treatment of injuries
induced by infection with influenza A H1N1.
Example 2. Induction of More Serious ALI by Influenza A Virus H1N1
in FGF-2 Deficient Mice
[0074] Each of 6 of 4-6 weeks old C57BL/6 mice (or 6 of 4-6 weeks
old FGF-2 gene knocked-out mice) was securely fixed, and
intraperitoneally injected with 50-60 .mu.L of 1 g/100 mL solution
of pentobarbital sodium by a 1 mL sterile syringe for anesthesia;
the anesthetized mice were kept with their head backward and upward
leaned to bring their nasal cavities into an upward position, a
virus solution of BJ501 strain (10.sup.5.5 TCID.sub.50/mouse) was
added dropwise to their nasal cavity at each side through a
pipette; the mice were kept in this posture for 15 seconds, to
allow the virus to enter into their respiratory tracts; 5 days
after infection with the influenza A virus, the mice were killed by
means of intraperitoneal injection of excessive anesthetic; three
of the killed mice were fixed on a small animal operating table,
and skin and bone of their chest were removed to expose thoracic
cavity, from which lung together with heart were removed, and
washed with sterile PBS buffer solution to wash off the blood on
the surface, and then placed into a paraformaldehyde fixing
solution for fixation at room temperature for 48 h, followed by a
series of treatments such as embedding, slicing, HE staining; the
other three of the killed mice were fixed on a small animal
operating table, and skin and bone of their chest were removed to
expose thoracic cavity, from which the entire lungs were taken off,
subjected to removal of surface blood and excessive connective
tissues, and weighed, and the wet weight of the lung was recorded;
then, the lungs were placed in a tissue drier at a high temperature
of 55.degree. C. and dried; after 24 h, the lungs were taken out
and weighed for dry weight when cooled to room temperature, so as
to obtain a wet/dry ratio=wet lung weight/dry lung weight.
[0075] The results of slice staining are shown in FIG. 2
(.times.200 folds), wherein A represents the lung tissue of a
C57BL/6 mouse, and B represents the lung tissue of a FGF-2 gene
knocked-out mouse. In the C57BL/6 mouse infected with influenza A
virus H1N1, serious pathological injuries occurred in lung tissue,
normal lung tissue structure was destroyed, and the lung markings
were disordered, accompanied with pathological injuries such as
bleeding, inflammatory effusion, as well as massive red cells,
inflammatory cell infiltration, and the like. The FGF-2 gene
knocked-out mouse infected with the same titer of virus had more
significant pathological injuries in lung tissue, and more
significant pathological changes such as bleeding, effusion,
inflammatory cell infiltration, or the like, unclear lung marking,
and non-intact structure.
[0076] The results of "wet/dry ratio" are shown in FIG. 3. The
"wet/dry ratio" of lung can reflect the extent of acute pulmonary
edema in a mouse. In a FGF-2 gene knocked-out mouse infected with
influenza A virus H1N1, the wet/dry ratio of lung was significantly
increased than that of a C57BL/6 mouse (*P<0.05), which
indicates that knockout of FGF-2 can deteriorate pulmonary edema in
a mouse infected with influenza A virus H1N1. The results indicate
that more serious ALI can be induced by influenza A virus H1N1 in a
FGF-2 deficient mouse.
Example 3. Induction of More Serious ALI by Influenza A Virus H1N1
in FGF-2 deficient Mice
[0077] Each of 20 of 4-6 weeks old C57BL/6 mice (or 20 of 4-6 weeks
old FGF-2 gene knocked-out mice) was securely fixed, and
intraperitoneally injected with 1 mL sterile syringe 50-60 .mu.L of
1 g/100 mL solution of pentobarbital sodium for anesthesia; the
anesthetized mice were kept with their head backward and upward
leaned to bring their nasal cavities into an upward position, 10
.mu.L virus solution of PR8 strain (10.sup.5.5 TCID.sub.50/mouse)
was added dropwise to their nasal cavity at each side through a
pipette; the mice were kept in this posture for 15 seconds, to
allow the virus to enter into their respiratory tracts; 14 of the
mice were subjected to survival statistics (the day before the
infection with influenza A virus was recorded as Day 0; from the
point of the infection with influenza A virus, 24 hours later was
recorded Day 1, and so on) and weight monitoring; the remaining 6
mice were killed by means of intraperitoneal injection of excessive
anesthetic 5 days after infection with influenza A virus; three of
the killed mice were fixed on a small animal operating table, and
skin and bone of their chest were removed to expose thoracic
cavity, from which lung together with heart were removed, and
washed with a sterile PBS buffer solution to wash off the blood on
the surface, and then placed into a paraformaldehyde fixing
solution for fixation at room temperature for 48 h, followed by a
series of treatments such as embedding, slicing, HE staining; the
other three of the killed mice were fixed on a small animal
operating table, and skin and bone of their chest were removed to
expose thoracic cavity, from which their entire lungs were taken
off, and subjected to removal of surface blood and excessive
connective tissues, weighed, and the wet weight of the lung was
recorded; then, the lungs were placed in a tissue drier at a high
temperature of 55.degree. C. and dried; after 24 h, the lungs were
taken out and weighed for dry weight when cooled to room
temperature, so as to obtain a wet/dry ratio=wet lung weight/dry
lung weight.
[0078] The statistics results of survival rate of mice infected
with influenza A virus are shown in FIG. 4. Infected with the same
titers of influenza A virus H1N1, the mortality of C57BL/6 mice was
significantly lower than the FGF-2 gene knocked-out mice
(*P<0.05). The statistical results of body weight change (i.e.,
a ratio of the body weight at a certain day to that at Day 0) of
mice infected with influenza A virus are shown in FIG. 5. Infected
with the same titers of influenza A virus H1N1, C57BL/6 mice had a
body weight change significantly less than that of the FGF-2 gene
knocked-out mice (*P<0.05). The results indicate that FGF-2
plays an essential role in the protection of mice infected with
influenza A virus H1N1 from death, and an intervention targeting
FGF-2 molecules may play an important role in the protection of
treating the one infected with influenza A virus H1N1.
[0079] The results of slice staining are shown in FIG. 6
(.times.200 folds), wherein A represents the lung tissue of a
C57BL/6 mouse, and B represents the lung tissue of a FGF-2 gene
knocked-out mouse. In the C57BL/6 mouse infected with influenza A
virus H1N1, serious pathological injuries occurred in lung tissue,
normal lung tissue structure was destroyed, and lung markings were
disordered, accompanied with pathological injuries such as
bleeding, inflammatory effusion, as well as massive red cells,
inflammatory cell infiltration, and the like. The FGF-2 gene
knocked-out mouse infected with the same titer of virus had more
significant pathological injuries in lung tissue, and more
significant pathological changes such as bleeding, effusion,
inflammatory cell infiltration, or the like, unclear lung markings,
and non-intact structure.
[0080] The results of "wet/dry ratio" are shown in FIG. 7. In a
FGF-2 gene knocked-out mouse infected with influenza A virus H1N1,
the wet/dry ratio of lung was significantly increased than that of
a C57BL/6 mouse (*P<0.05), which indicates that knockout of
FGF-2 can deteriorate pulmonary edema in a mouse infected with
influenza A virus H1N1. These results indicate that more serious
ALI can be induced by influenza A virus H1N1 in a FGF-2 deficient
mouse.
Example 4. Ability of FGF-2 Recombinant Protein to Mitigate Acute
Lung Injury Induced by Infection with Influenza A Virus
[0081] I. Experiment I
[0082] For a experimental group (10 of 4-6 weeks old C57BL/6 mice):
at Day 1, each of the mice was intravenously injected with 100
.mu.l of a solution of FGF-2 recombinant protein (at a protein
concentration of 0.5 mg/ml); at Day 2, each of the mice was
securely fixed, and intraperitoneally injected with 50-60 .mu.L of
1 g/100 mL solution of pentobarbital sodium by a 1 mL sterile
syringe for anesthesia; the anesthetized mice were kept with their
head backward and upward leaned to bring their nasal cavities into
an upward position, and 10 .mu.L virus solution of PR8 strain
(10.sup.5.5 TCID.sub.50/mouse) was added dropwise to their nasal
cavity at each side through a pipette; the mice were kept in this
posture for 15 seconds to allow the virus to enter into their
respiratory tract; at Day 3, each of the mice was intravenously
injected with 100 .mu.l of the FGF-2 recombinant protein solution
(at a protein concentration of 0.5 mg/ml); at Day 5 , each of the
mice was intravenously injected with 100 .mu.l of the FGF-2
recombinant protein solution (at a protein concentration of 0.5
mg/ml); the survival of the mice was determined daily.
[0083] For a control group (10 of 4-6 weeks old C57 BL/6 mice): the
mice were treated in the same way except that an equal volume of
PBS buffer was used instead of the FGF-2 recombinant protein
solution.
[0084] The statistic results of the survival rate from Day 1 to Day
14 are shown in FIG. 8. After infected with the same titer of
influenza A virus H1N1, the control group had a mortality
significantly higher than that of the mice of the experimental
group (*P<0.05). The results indicate an important therapeutic
effect of FGF-2 in death of the mice infected with influenza A
virus H1N1, and an intervention targeting FGF-2 molecules may take
an essential part in the recovery of treating the one infected with
influenza A virus H1N1.
[0085] II. Experiment II
[0086] For a experimental group (6 of 4-6 weeks old C57BL/6 mice):
at Day 1, each of the mice was intravenously injected with 100
.mu.l of a solution of FGF-2 recombinant protein (at a protein
concentration of 0.5 mg/ml); at Day 2, each of the mice was
securely fixed, and intraperitoneally injected with 50-60 .mu.L of
1 g/100 mL solution of pentobarbital sodium by a 1 mL sterile
syringe for anesthesia; the anesthetized mice were kept with their
head backward and upward leaned to bring their nasal cavities into
an upward position, and 10 .mu.L virus solution of PR8 strain
(10.sup.5.5 TCID.sub.50/mouse) was added dropwise to their nasal
cavity at each side through a pipette; the mice were kept in this
posture for 15 seconds to allow the virus to enter into their
respiratory tract; at Day 3, each of the mice was intravenously
injected with 100 .mu.l of the FGF-2 recombinant protein solution
(at a protein concentration of 0.5 mg/ml), at Day 5 , each of the
mice was intravenously injected with 100 .mu.l of the FGF-2
recombinant protein solution (at a protein concentration of 0.5
mg/ml), at Day 6, the mice were killed by means of intraperitoneal
injection of excessive anesthetic; three of the killed mice were
fixed on a small animal operating table, and skin and bone of their
chest were removed to expose thoracic cavity, from which lung
together with heart were removed, and washed with a sterile PBS
buffer solution to wash off the blood on the surface, and then
placed into a paraformaldehyde fixing solution for fixation at room
temperature for 48 h, followed by a series of treatments such as
embedding, slicing, HE staining; the other three of the killed mice
were fixed on a small animal operating table, and skin and bone of
their chest were removed to expose thoracic cavity, from which
their entire lungs were taken off, and subjected to removal of
surface blood and excessive connective tissues, weighed and the wet
weight of the lung was recorded; then, the lungs were placed in a
tissue drier at a high temperature of 55.degree. C. and dried;
after 24 h, the lungs were taken out and weighed for dry weight
when cooled to the room temperature, so as to obtain a wet/dry
ratio=wet lung weight/dry lung weight.
[0087] For the control group (6 of 4-6 weeks old C57 BL/6 mice):
the mice were similarly treated except that an equal volume of PBS
buffer was used instead of the FGF-2 recombinant protein
solution.
[0088] The results of slice staining are shown in FIG. 9
(.times.200 folds), wherein A represents the lung tissue of a mouse
of the control group, and B represents the lung tissue of a mouse
of the experimental group. In the mouse of the control group
infected with influenza A virus H1N1, serious pathological injuries
occurred in lung tissue, normal lung tissue structure was
destroyed, and lung markings were disordered, accompanied with
pathological injuries such as bleeding, inflammatory effusion, as
well as massive red cells, inflammatory cell infiltration, and the
like. However, in the mouse of the experimental group infected with
the same titer of virus, neither significant pathological injury of
lung tissue, nor significant pathological changes such as bleeding,
effusion, inflammatory cell infiltration, or the like, was
observed, and their lungs were observed having clear markings and
perfect structure. The results indicate that FGF-2 plays an
important role in the protection of a mouse from acute pathological
injury of lung tissue induced by infection with influenza A virus
H1N1.
[0089] The results of wet/dry ratio are shown in FIG. 10. The mice
of the experimental group, after being infected with influenza A
virus H1N1, had a wet/dry ratio of lung significant lower than that
of mice of the control group (*P<0.05), which indicates that
FGF-2 can substantially mitigate pulmonary edema in a mouse induced
by infection with influenza A virus H1N1. The results suggest that
FGF-2 plays an important role in the protection from acute
pathological injury of lung tissue induced by infection with
influenza virus A.
Example 5. Ability of FGF-2 Recombinant Protein to Mitigate
Pathological Injury of Mouse Lung Tissue After Co-infection with
LPS and Zymosan A
[0090] For Group 1 (4 of 4-6 weeks old C57BL/6 mice): each of the
mice were intravenously injected with 100 .mu.l of a FGF-2
recombinant protein solution (at a protein content of 50 .mu.g) 12
hours prior to LPS infection, 1 hours prior to LPS infection, and 8
hours after LPS infection, respectively.
[0091] Infection: each of the mice was intraperitoneally injected
with 50-60 .mu.L of a 1 g/100 mL solution of pentobarbital sodium
by a 1 mL sterile syringe for anesthesia; the anesthetized mice
were kept with their head backward and upward leaned to bring their
nasal cavities into an upward position, and intranasally and
dropwise administered with 50 .mu.L of a LPS solution (100 .mu.g
LPS in a solvent of PBS buffer); the mice were kept in this posture
for 5 minutes to allow the LPS to enter into their respiratory
tracts; 1 hour after the infection with LPS, each of the mice was
intraperitoneally injected with 50-60 .mu.L of the 1 g/100 mL
pentobarbital sodium solution by a 1 mL sterile syringe for
anesthesia; the anesthetized mice were kept with their head
backward and upward leaned to bring their nasal cavities into an
upward position, and intranasally and dropwise administered with 50
.mu.L of a zymosan A solution (60 .mu.g LPS in a solvent of PBS
buffer); the mice were kept in this posture for 5 minutes, to allow
the LPS to enter into their respiratory tracts.
[0092] At 24 hours after the LPS infection, the mice were killed by
means of intraperitoneal injection of excessive anesthetic, and
fixed on a small animal operating table, and skin and bone of their
chest were removed to expose thoracic cavity, from which lung
together with heart were removed, and washed with a sterile PBS
buffer solution to wash off the blood on the surface, and then
placed into a paraformaldehyde fixing solution for fixation at room
temperature for 48 h, followed by a series of treatments such as
embedding, slicing, HE staining, etc.
[0093] Group 2: the mice were treated in the same way as Group 1
except for no injection of FGF-2 recombinant protein solution 12
hours prior to LPS infection, 1 hours prior to LPS infection, and 8
hours after LPS infection.
[0094] Group 3: the mice were treated in the same way as Group 1
except that an equal volume of PBS buffer solution was used instead
of the LPS solution, and an equal volume of PBS buffer solution was
used instead of the zymosan A solution.
[0095] The results are shown in FIG. 11, wherein A represents Group
1, B represents Group 2, and C represents Group 3. No significant
pathological injury and pathological changes such as bleeding,
effusion, inflammatory cell infiltration, or the like, but clear
lung markings and perfect structure were observed in the lung
tissue of the mice of Group 3. Significant pathological injury and
pathological changes such as bleeding, effusion, inflammatory cell
infiltration, or the like, unclear lung markings, and non-intact
structure were observed in the lung tissue of the mice of Group 2.
No significant pathological injury and pathological changes such as
bleeding, effusion, inflammatory cell infiltration, or the like,
but clear lung markings and perfect structure were observed in the
lung tissue of the mice of Group 1. The results indicate that FGF-2
plays an important protective role in acute pathological injuries
of lung tissue in mouse induced by co-infection with LPS and
zymosan A.
INDUSTRIAL APPLICATION
[0096] The present invention discloses uses of FGF-2 in the
manufacture of a drug for treating and/or preventing lung injury,
for preventing and/or treating influenza, and for preventing and/or
treating a disease induced by an influenza virus. The present
invention may be valuable for the treatment and prevention of above
diseases.
Sequence CWU 1
1
21155PRTHomo sapiens 1Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala
Leu Pro Glu Asp Gly 1 5 10 15 Gly Ser Gly Ala Phe Pro Pro Gly His
Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe
Phe Leu Arg Ile His Pro Asp Gly Arg 35 40 45 Val Asp Gly Val Arg
Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu 50 55 60 Gln Ala Glu
Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 65 70 75 80 Arg
Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90
95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr
100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala
Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly
Pro Gly Gln Lys 130 135 140 Ala Ile Leu Phe Leu Pro Met Ser Ala Lys
Ser 145 150 155 2468DNAHomo sapiens 2atggcagccg ggagcatcac
cacgctgccc gccttgcccg aggatggcgg cagcggcgcc 60ttcccgcccg gccacttcaa
ggaccccaag cggctgtact gcaaaaacgg gggcttcttc 120ctgcgcatcc
accccgacgg ccgagttgac ggggtccggg agaagagcga ccctcacatc
180aagctacaac ttcaagcaga agagagagga gttgtgtcta tcaaaggagt
gtgtgctaac 240cgttacctgg ctatgaagga agatggaaga ttactggctt
ctaaatgtgt tacggatgag 300tgtttctttt ttgaacgatt ggaatctaat
aactacaata cttaccggtc aaggaaatac 360accagttggt atgtggcact
gaaacgaact gggcagtata aacttggatc caaaacagga 420cctgggcaga
aagctatact ttttcttcca atgtctgcta agagctga 468
* * * * *
References